Human papillomaviruses (HPVs) are the major cause of several cancers including cervical cancer, a leading cancer in women worldwide. Most infections are self limiting, but the sheer number of infections makes HPV-induced cancers very common. HPV infections can last for decades and cannot be cleared by currently available prophylactic vaccines. For this reason, understanding how HPV persistence is maintained in infected tissues is the critical next step in managing HPV-related diseases. Although HPVs infect epithelial keratinocytes exclusively, other cells in the stromal microenvironment, including fibroblasts, endothelial cells, and leukocytes, provide growth regulatory and innate immune signals that potentially affect the course of persistent HPV infection. We have found that the HPV oncoproteins E5 and E7 regulate overlapping sets of cellular factors that signal to stromal cells, most prominently the type I IFN and TGF pathways. This means that the virus can manipulate not just infected cells but also neighboring cells of the stromal microenvironment. We hypothesize that communication with stromal fibroblasts through the IFN and TGF pathways is crucial to the HPV life cycle. In this proposal we will determine 1) the importance of TGF and IFN signaling in stromal fibroblasts for the HPV life cycle. 2) How TGF and IFN signaling in keratinocytes impacts viral replication. 3) The molecular mechanism for regulation of IFNs and TGF by E5. The strength of this proposal is that it is the first to systematically investigate the role of the stromal microenvironment in the normal, evolved HPV life cycle and to focus on TGF and IFN in their functional setting as paracrine communication systems in the context of HPV infection. We will employ HPV replication in a three dimensional organotypic culture system as a functional readout in order to ensure our findings have relevance to the evolved HPV life cycle. At the end of this work, we will possess a more complete understanding of the interrelationship between innate immunity and growth factor signaling, and will thus gain insights into the problem of HPV persistence, as well as other conditions such as cancer, inflammatory diseases, and infections by other viruses.